Bilirubin is a breakdown product of the heme moiety of hemoglobin. Two forms of bilirubin have been distinguished in biological fluids. "Free" or "unconjugated" bilirubin is transported in plasma by attachment to proteins, primarily albumin. In the hepatic cells, free bilirubin is conjugated with glucuronyl or sulfate radicals to form "conjugated" bilirubin. Conjugated bilirubin is freely soluble in water, while unconjugated bilirubin is extremely insoluble in water.
Elevated levels of conjugated bilirubin are usually found in serum specimens derived from patients with obstructive jaundice; elevated levels of unconjugated bilirubin are usually found in serum specimens derived from patients with hemolytic jaundice. In the diagnosis of these disease states, both the absolute concentrations of conjugated and unconjugated bilirubin and their relative concentrations are of importance. Therefore, there is great need for simple, accurate and reproducible methods for the determination of both types of bilirubin. It may be noted that determination of only one of the types of bilirubin may be sufficient after their ratio has initially been determined for a particular patient.
Bilirubin is sold commercially both for use in standardizing procedures for determining bilirubin in biological samples and for use in research. Bilirubin determination methods are also needed in the preparation of this commercial product.
Bilirubin reacts with diazobenzene-p-sulfonic acid to form two isomeric azopigments of identical light absorption properties. Commonly, these two isomeric azopigments are collectively referred to as "azobilirubin."
Conjugated and unconjugated bilirubin differ considerably in their reaction behavior with diazobenzene-p-sulfonic acid. Conjugated bilirubin reacts directly and immediately with diazobenzene-p-sulfonic acid at an acid pH of about 1.5 to 2.0. Unconjugated bilirubin reacts with diazobenzene-p-sulfonic acid only in the presence of an accelerating agent. As a result, conjugated bilirubin is also referred to as "direct bilirubin" and unconjugated bilirubin as "indirect bilirubin."
A number of methods have been devised for the estimation of bilirubin based on the diazo reaction of bilirubin with diazobenzene-p-sulfonic acid. In these methods direct bilirubin is determined in the absence of an accelerating agent, and total bilirubin (i.e., the sum of direct and indirect bilirubin) is determined in the presence of an accelerating agent. These methods differ from each other in a number of details. Various accelerating agents have been employed, such as methanol, acetic acid, urea or a caffeine-sodium benzoate-sodium acetate reagent. Also, the pH of the ultimate reaction mixture has been varied. Azobilirubin is blue in strong acid or alkaline medium, and pink or red in neutral medium. One widely used procedure (Malloy and Evelyn, J. Bio. Chem. 119, pages 481-490 (1937), determines the pink color of azobilirubin in a neutral ultimate reaction mixture. Numerous modifications of this method have also been used. Other methods (such as Jendrassik and Grof, Biochemische Zeitschrift, 297, pages 81-89 (1938) measure the blue color of azobilirubin in alkaline medium. The alkaline azobilirubin methods have certain advantages: they are more sensitive because of the intensity of the blue azobilirubin color; they avoid protein precipitation and turbidity in the ultimate reaction mixture; and they are free from interference by hemoglobin and other colored substances usually found in biological fluids.
In the estimation of bilirubin by the alkaline azo method, it is customary to add ascorbic acid to the reaction mixture at a certain time after the addition of diazo reagent. Added ascorbic acid in the reaction mixture destroys unreacted diazobenzene-p-sulfonic acid in the reaction mixture, thereby preventing secondary reactions with indirect bilirubin in the "direct bilirubin" reaction mixture when alkali is added. In both the "direct bilirubin" and "total bilirubin" reactions, it also stabilizes the blue color of azobilirubin in the ultimate alkaline reaction mixture.
Although bilirubin has been determined by a diazo reaction for over fifty years, all of the azo methods have certain drawbacks. One of the chief drawbacks is the instability of reagents utilized in the reaction. The diazo reagent, comprising diazobenzene-p-sulfonic acid in an acidic solution, was long considered stable only for a matter of minutes, although more recently it has been reported that the reagent is essentially stable for twenty-four hours. The procedure for the preparation of fresh diazo reagent is to mix an aliquot of an aqueous solution of sodium nitrite with an aliquot of an acidic solution of sulfanilic acid. These solutions are also considered to have limited stability in storage.
To simplify the preparation of diazobenzene-p-sulfonic acid without using solutions of sulfanilic acid and sodium nitrite, Sherman and Zak, Am. J. Clin. Pathol., 23 946 (1953) introduced a diazo tablet which consisted of one hundred milligrams of sodium sulfanilate and twenty milligrams of sodium nitrite. Prior to running the diazo reaction, they dissolved this tablet in an aliquot of hydrochloric acid of know concentration to obtain the diazo solution. This has a number of problems. When the tablet is dissolved in hydrochloric acid, the sodium nitrite dissolves quickly to form nitrous acid, much of which decomposes before it can react completely with the essentially insoluble sulfanilic acid. The inhomogeneity of the tablet also contributes to variations in the final concentration of diazo compound formed from the tablet. The diazo solution derived from the diazo tablet is sometimes yellow and not suitable for use in the diazo reaction, and the shelf life of the diazo tablet is not well established.
To avoid the problems caused by the instability of diazobenzene-p-sulfonic acid and of the reagents used to prepare it, more stable diazo reagents have been prepared from various aromatic amines. The light absorption properties of isomeric azobilirubins derived from these diazo reagents have not yet been established, however. More importantly, by use of these diazo reagents it has not been possible to estimate direct bilirubin accurately and reproducibly, or in a manner which correlates well with the results obtained with diazobenzene-p-sulfonic acid.
Solutions of ascorbic acid are also known to be highly unstable. Therefore, the use of a freshly prepared solution of ascorbic acid has been recommended in the alkaline azo method of determining bilirubin. The ascorbic acid is also rapidly destroyed in the strongly alkaline ultimate solution, and therefore does not offer long-lasting protection of the blue azobilirubin color.
Summaries of various azobilirubin determinations are set out in R. J. Henry, Clinical Chemistry: Principles and Technics (Hoeber Medical Division, 1964), pages 572-594; S. R. Gambino and J. Di Re, Bilirubin Assay (American Society of Clinical Pathologists, 1968); and Denney et al U.S. Pat. No. 3,652,222 (1972), all of which are hereby incorporated by reference.